There are currently three research projects in the laboratory.

Microbe Source Tracking.

Little is known about the fate of microbes released into the environment. How long do bacteria survive? What physiological changes do they undergo over time? How far and how fast do they travel through soils? Current methods to monitor changes in population profiles cannot answer these questions. Our research is attempting to monitor individual cells temporally and spatially to examine the movement, longevity, and physiological status of bacteria in the environment. This field is called bacterial, or microbe, source tracking (BST/MST). We are in the process of developing a BST method that can be used to identify the source of fecal contaminants in surface waters. In a pilot study awaiting final approval from the US EPA, a gfp-tagged natural E. coli isolate will be introduced into the subsurface area adjacent to a stream on campus. Samples collected from monitoring wells will be analyzed via PCR and microscopy for the presence of dead, culturable, and viable-but-nonculturable forms of the GMO. A patent on this BST method is currently under review and a biotechnology company, BioTrackers, Inc., was formed to explore the commercial applications of this method. The U.S. EPA has supported this research through SBIR funding.

Change in microbial diversity in CF patients in response to antibiotic treatment.

Cystic Fibrosis (CF) affects the epithelial cells of mucus membranes in the human body. In the lungs, the mutation in the CFTR gene results in the production of thick, sticky mucus that becomes difficult to move out of the airways.  Bacteria that are normally not pathogenic in the lungs grow in this mucus and cause chronic infections. In CF patients, there are several bacteria that are resistant to most known antibiotics, making treatment difficult. Bacteria in CF patients are usually characterized in the clinic by cultivation techniques, but culturing only identified a fraction of bacteria present.

New developments in sequencing technology are revolutionizing the study of complex microbial communities. The goal of this project is to provide a detailed mechanistic view of how antibiotic resistance is developed in CF patients and will serve as a foundation for the development of personalized courses of treatment to manage opportunistic infections. Two techniques being used are pyrosequencing and ARISA.  

In an ARISA experiment, DNA is extracted from a sputum sample and subjected to PCR using primers from conserved 16S and 23S regions that are separated by an intergenic region whose length varies in different bacteria/taxa. The length of this intergenic sequence (IGS) from organisms present in a sample is determined and the thousands of IGS lengths from a given sample are compiled into a single profile. An ARISA profile, then, is a “signature” of the microbial community with each peak representing a distinct microbial taxon.

In preliminary experiments analyzing sputum samples from a CF patient via ARISA and pyrosequencing. These data indicate that 1) bacteria diversity can be high in CF patients not having an active infection, 2) bacterial diversity correlates with antibiotic treatment, 3) during standard antibiotic treatment, bacteria persist in the lung and 4) microbial diversity may increase during an active infection.

Mammalian host genotype affects on gastrointestinal tract microbiome.

This project has as its goal determining if the mammalian host influences the diversity and type of fitness-promoting bacteria found in the gut. The mammalian gastrointestinal tract is colonized with vast numbers of microbes representing many different species belonging to a limited number of broad taxonomic divisions. These microbes aid in digestion, promote immune system development, and provide a barrier to pathogen invasion.  Despite the considerable recent progress in the description of microbial symbiotic communities, the specific molecular interactions between host and microbe, and the contributions of individual microbial taxa and genes to host phenotypes, remain largely unknown.

In results published by our collaborators, Drs. Anthony Fodor (Department of Bioinformatics and Genomics) and Timothy Lightfoot (Department of Kinesiology), one strain of mouse (“129”) has a lower level of physical activity than does another strain (“SM/J”). Examining the gut flora via ARISA indicates that these strains have distinct microbial communities. The hypotheses are that 1) host activity phenotypes can also be linked to, and transferred by, the gut microbial community, and that 2) the host genotype influences the composition of the gut microbiota. Collaborative experiments are under way to monitor changes in gut biota ARISA profiles as the mouse flora is manipulated via antibiotic treatment and bacterial reinoculation, and to identify the fitness-promoting bacterial species.

Yankson, K., and Steck, T.R. A Strategy for Extracting DNA from Clay Soil, and Detecting a Specific Target Sequence via Selective Enrichment and Real-Time (quantitative) PCR Amplification. 2009. Appl. Environ. Microbiol. 75(18):xxx-xxx (in press).

Anderson, M., Bollinger, D., Hagler, A., Hartwell, H., Rivers, B., Ward, K., and Steck, T. R. 2004. Viable but nonculturable bacteria are present in mouse and human urine specimens. J. Clin. Microbiol. 42(2):753-758

Grey, B., and Steck, T.R. 2001. Concentrations of copper thought to be toxic to Escherichia coli can induce the viable but nonculturable condition. Appl. Environ. Microbiol. 67(11):5325-5327.

Grey, B., and Steck, T.R. 2001. The viable but non-culturable state of Ralstonia solanacearum may be involved in long-term survival and plant infection. Appl. Environ. Microbiol. 67(9):3866-3872. 

Rivers, B., and Steck, T.R. 2001. Viable but nonculturable uropathogenic bacteria are present in the mouse urinary tract following urinary tract infection and antibiotic therapy. Urol. Res. 29:60-66. 

Ghezzi, J., and Steck, T.R. 1999. Induction of the viable but nonculturable condition in Xanthomonas campestris pv. campestris in liquid microcosms and sterile soil. FEMS Microbiol. Ecol. 30(3):203-208. 

Deepika Gaddam. M.S. student. Research project - development of a new bacterial source tracking method.

Joshua Stokell. M.S. student. Research project - changes in microbial diversity in CF patients in response to antibiotic therapy.